ultrastructural localization of g-proteins and the channel protein … · 2013. 11. 26. ·...

Ultrastructural Localization of G-Proteins and the Channel Protein TRP2

to Microvilli of Rat VomeronasalReceptor Cells

BERT PH. M. MENCO,1* VIRGINIA McM. CARR,1 PATRICK I. EZEH,1

EMILY R. LIMAN,2AND MAYA P. YANKOVA1

1Department of Neurobiology and Physiology, Northwestern University, Evanston,Illinois 60208-3520

2Department of Biological Sciences/Neurobiology, University of Southern California,Los Angeles, California 90089-2520

ABSTRACTMicrovilli of vomeronasal organ (VNO) sensory epithelium receptor cells project into the

VNO lumen. This lumen is continuous with the outside environment. Therefore, the microvilliare believed to be the subcellular sites of VNO receptor cells that interact with incomingVNO-targeted odors, including pheromones. Candidate molecules, which are implicated in VNOsignaling cascades, are shown to be present in VNO receptor cells. However, ultrastructuralevidence that such molecules are localized within the microvilli is sparse. The present studyprovides firm evidence that immunoreactivity for several candidate VNO signaling molecules,notably the G-protein subunits Gia2 and Goa, and the transient receptor potential channel 2(TRP2), is localized prominently and selectively in VNO receptor cell microvilli. Although Gia2and Goa are localized separately in the microvilli of two cell types that are otherwise indistin-guishable in their apical and microvillar morphology, the microvilli of both cell types areTRP2(1). VNO topographical distinctions were also apparent. Centrally within the VNO sensoryepithelium, the numbers of receptor cells with Gia2(1) and Goa(1) microvilli were equal. How-ever, near the sensory/non-sensory border, cells with Gia2(1) microvilli predominated. Scatteredciliated cells in this transition zone resembled neither VNO nor main olfactory organ (MO)receptor cells and may represent the same ciliated cells as those found in the non-sensory part ofthe VNO. Thus, this study shows that, analogous to the cilia of MO receptor cells, microvilli ofVNO receptor cells are enriched selectively in proteins involved putatively in signal transduction.This provides important support for the role of these molecules in VNO signaling. J. Comp.Neurol. 438:468–489, 2001. © 2001 Wiley-Liss, Inc.

Indexing terms: immunolocalization; freeze substitution; olfaction; signal transduction; Gia2; Goa;

transient receptor potential channel

In several classes of vertebrates and insects, there aretwo olfactory pathways that play different but overlappingroles. One of these detects predominantly gender and con-specific exuded odors, whereas the other detects moregeneral odors (Hildebrand and Shepherd, 1997; Johnston2000). In vertebrates there is some overlap between theperceptive modalities of main (MO) and vomeronasal ol-factory organs (VNO; Johnston, 2000). However, it is as-sumed that in most vertebrates, the MO has evolved todetect general odors whereas the VNO is specialized forendocrine responses to odors and prey recognition, thelatter especially in some reptiles (Halpern, 1987; Keverne,1999; Dulac, 2000; Johnston, 2000).

The signaling pathways of MO and VNO are parallel,but individual components differ (Berghard et al., 1996;Wu et al., 1996; Tirindelli et al., 1998). Although a subset

Grant sponsor: NIH-NIDCD; Grant number: DC02491 (BPhMM); Grantnumber: DC02774 (VMcMC); Grant number: DC04564 (ERL); Grant spon-sor: NSF; Grant number: IBN-0094709 (BPhMM).

*Correspondence to: Dr. Bert Ph. M. Menco, Department of Neurobiologyand Physiology, O.T. Hogan Hall, 2153 North Campus Drive, Northwest-ern University, Evanston, IL 60208-3520.E-mail: [email protected]

Received 28 November 2000; Revised 19 April 2001; Accepted 4 July2001

THE JOURNAL OF COMPARATIVE NEUROLOGY 438:468–489 (2001)

© 2001 WILEY-LISS, INC.

of MO receptor cells may make use of a different cascade(Meyer et al., 2000), the signaling proteins that are acti-vated sequentially by the interaction between odors andodorant receptors are the same for the majority of receptorcells. These proteins include the specific GTP-binding (orG)-protein Golf, type III adenylyl cyclase (AC), and olfac-tory cyclic nucleotide gated channels (OCNCs; Buck,2000). All of these molecules are particularly abundant inspecialized cilia (Menco, 1997a; Menco and Morrison,2002).

VNO receptor cells bear, for still unclear reasons, mi-crovilli instead of cilia (for rat: Vaccarezza et al., 1981).Also differing from the situation in the MO are resultsfrom immunocytochemical studies on G-proteins thatshowed that rodents have two major populations of VNOreceptor cells that each makes use of a different signalingcascade. One cell type expresses a-subunits of the hetero-trimeric G-protein Gi2, the other a-subunits of Go (Jia andHalpern, 1996). Both of these are fairly closely related(Fig. 1 on p. 297 in Watson and Arkinstall, 1994), arepertussis-toxin sensitive, and may play a role in a phos-pholipase C (PLC)-dependent phosphoinositide hydrolysis(Watson and Arkinstall, 1994), also in the VNO (Dulac,2000; Holy et al., 2000). Immunocytochemistry showed theGia2(1) and Goa(1) cells to be evenly distributed along the

length of the VNO neuroepithelium. However, somata ofGia2-expressing cells are localized more superficially inthe VNO neuroepithelium than somata of Goa-expressingcells (Jia and Halpern, 1996), a finding confirmed by insitu hybridization (Berghard and Buck, 1996). Light mi-croscopic (LM) studies with different antibodies furtherdemonstrated that immunoreactivity (IR) for both Gia2and Goa occurs at the luminal edge of the epithelium,which is consistent with antigen localization in receptorcell apices and microvilli (Berghard and Buck, 1996).There is a correlation between the expression of each ofthe VNO G-proteins and two heptahelical odorant super-families, V1R for Gia2 and V2R for Goa (Herrada andDulac, 1997; Matsunami and Buck, 1997; Ryba and Tir-indelli, 1997). (A third family, V3R [Pantages and Dulac,2000], appears to be a group within the V1R superfamily[Mombaerts, 2001].) Furthermore, in situ hybridizationdemonstrated that a member of the transient receptorpotential (TRP) channel family, TRP2, is amply present inboth Gia2(1) and Goa(1) cells. Immunocytochemistry inisolated single rat VNO receptor cells demonstrated thatTRP2 and Gia2 are highly restricted in what are presumedto be receptor cell microvillar structures. Therefore, it wasproposed that TRP2 is a likely candidate for current gen-eration in VNO receptor cells (Liman et al., 1999). This

TABLE 1. Antibodies Tested for Their Fine Structural Presence in VNO Tissues1

Antibodies Supplier Catalog no. PeptidePolyclonal ormonoclonal Peptide sequence

Gia12 Santa Cruz

Biotechnology3I-20, sc-391 93-112 Polyclonal Highly divergent internal region,

IDFGDSARADDARLQLFVLAGGia1-3

2 Santa CruzBiotechnology

C-10, sc-262 345-354 Polyclonal C-terminal, KNNLKECGLY

Gia1,22 Calbiochem4 371723-S 245-254 Polyclonal C-terminal, KNNLKDCGLF

Goa2 Santa Cruz

BiotechnologyK-20, sc-387 105-124 Polyclonal Divergent internal region,

KMVCDVVSRMEDTEPFSAELTRP22 Fusion protein as described by Liman et al. (1999) Polyclonal

1Antibodies that made a case for VNO signaling at the level of receptor cell microvilli are listed. Blocking controls, using the antigenic peptides, were performed for the antibodieslisted here.2Antibodies affinity purified.3Santa Cruz, CA.4La Jolla, CA.

TABLE 2. Antibodies Tested for Their Fine Structural Presence in VNO Tissues1

Antibodies Supplier Catalog no.Polyclonal ormonoclonal

Gia22 Chemicon3 MAB3077 Monoclonal

Goa4 NEN5 NEI-804 Polyclonal

Gsa4 NEN6 NEI-805 Polyclonal

Gsa2 Calbiochem7 372732-S Polyclonal

Golfa2 Jones and Reed (1989)6,8 DJ6.3AP Polyclonal

Gaq/114 NEN NEI-809 Polyclonal

Gaq/112 Santa Cruz Biotechnology9 C-19, sc-392 Polyclonal

Type II adenylyl cyclase2 Santa Cruz Biotechnology C-20, sc-587 PolyclonalType III adenylyl cyclase2 Bakalyar and Reed (1990)6,8 HAB-1 PolyclonalOCNC1 (subunit 1, olfactory cyclic nucleotide gated channel)2 Matsuzaki et al. (1999)6,10 PolyclonalIP3 (inositol 1,4,5 trisphosphate) receptor4 See Kalinoski et al. (1994)11 PolyclonalOlfactory marker protein4 Margolis (1982)12 Polyclonal

1Antibodies that gave ambiguous or negative results in the VNO are listed. These include antibodies to MO signaling proteins (see footnote 6). Blocking controls, using the antigenicpeptides, were only performed for the antibodies in Table 1.2Antibodies affinity purified.3Temecula, CA. This antibody labeled dendritic endings and microvilli of VNO receptor cells in a fine structural study that made use of pre-embedding immunocytochemistry(Matsuoka et al., 2001).4Not purified antisera.5Boston, MA.6Same as used in the MO. Labeled predominantly olfactory receptor cell cilia (Menco et al., 1994; Menco, 1997a; Matsuzaki et al., 1999).7La Jolla, CA.8Gift of Dr. R.R. Reed, Johns Hopkins University, Baltimore, MD.9Santa Cruz, CA.10Gift of Dr. G.V. Ronnett, Johns Hopkins University, Baltimore, MD.11Gift of Dr. D.L. Kalinoski, University of Miami, FL.12Gift of Dr. F.L. Margolis, University of Maryland, Baltimore, MD. This antibody worked well in an other fine structural study, but that study made use of pre-embeddingimmunocytochemistry (Johnson et al., 1993). For MO, see Menco (1994).

469G-PROTEINS AND TRP2 IN VOMERONASAL MICROVILLI

channel may be active in the VNO across a wide variety ofspecies, including some reptiles (Murphy et al., 2001).

V1R(1)/Gia2(1) and V2R(1)/Goa(1) VNO receptor cellsproject to a distinct region of the accessory olfactory bulb(AOB), the VNO’s first relay station in the brain. V1R(1)/Gia2(1) cells project anteriorly and V2R(1)/Goa(1) cellsproject posteriorly (Berghard and Buck, 1996; Jia andHalpern, 1996; Herrada and Dulac, 1997; Matsunami andBuck, 1997; Ryba and Tirindelli, 1997; Sugai et al., 1997;Halpern et al., 1998; Wekesa and Anholt, 1999). TheV1R(1)/Gia2(1) signaling cascade may be involved in therecognition of gender-specific volatile pheromones in ro-dents. The V2R(1)/Goa(1) cascade may subserve recogni-tion of conspecific nonvolatile proteinaceous compoundsthat need not be gender or strain specific (Cavaggioni etal., 1999; Inamura et al., 1999; Keverne, 1999; Krieger etal., 1999; Dulac, 2000).

Fine structural (immuno)cytochemical characteriza-tions of VNO receptor cells have shown that apices andmicrovilli of rat VNO receptor cells contain Gia2 and Goa

(Matsuoka et al., 2001) and, in a subset of these cells,

putative receptor VN6 (Takigami et al., 1999). mRNA foranother VNO receptor, V2R-8, has been localized in mi-crovillous receptor cells in the gold fish (Anderson et al.,1999). Optimally preserving tissue antigenicity, by usingfreeze-substitution electron microscopy (EM), combinedwith postembedding immunogold immunocytochemistry(Gilkey, 1993; Griffiths, 1993; Menco, 1995), this studydemonstrates that Gia2, Goa, and TRP2 are expressedmuch more abundantly in the VNO sensory microvillithan in other parts of the VNO receptor cell apices. More-over, at the level of these microvilli, each of these proteinshas its own peculiar distribution across the epithelial sur-face (preliminary reports: Menco, 1997b; Menco andYankova, 1999).

MATERIALS AND METHODS

Animals

Forty adult male Sprague Dawley rats (Harlan, India-napolis, IN), 2–3 months old, were used in this study. All

Fig. 1. Immunoblots for affinity-purified antibodies to Goa (A),Gia1,2 (B), Gia1-3 (C), and Gia1 (D) in pellet (P) and supernatant (S)membrane preparations of rat VNO epithelia. Antibodies to Goa, Gia1,and Gia1-3 were used at dilutions of 1:200 and antibodies to Gia1,2 at adilution of 1:1,000. Bands of appropriate molecular weight (kDa) are

marked with an arrow. All antibodies, apart from those to Gia1,labeled proteins of the appropriate weights. For S, the listed aliquotscorrespond to 13 mg (4 ml) to 52 mg (16 ml). For P, they correspond to2.5 mg (2 ml) to 27.5 mg (22 ml).

470 B.Ph.M. MENCO ET AL.

Fig. 2. Frontal sections through VNO showing sensory and non-sensory epithelia (s and ns in A and D). Sensory surfaces demonstrateIR for purified rabbit polyclonal antibodies to Gia1-3 (A,D; 1/1,000),Gia1,2 (B,E; 1/100), and Goa (C,F; 1/1,000) (arrows). For controls, PBSwas used instead of primary antibodies (G,H). I: Schematic map thatlinks the LM survey presented here with the fine structural topo-graphical part of the study (Figs. 6A,B, 8-10; the manner in which theregions are numbered is explained in the Materials and Methods).Immunoabsorption with excess antigenic peptide suppressed the la-beling in all cases (Figs. 7A,B). The boxed areas in A-C and G arepresented at higher magnifications in D-F and H. VNO sensory sur-

face IR was more intense for Gia1-3 (A,D) than for Gia1,2 (B,E) and forGoa (C,F). Despite this, we elected to use the Gia1,2 antibodies ratherthan those to Gia1-3 for most of the study to demonstrate Gia2(1) cellsbecause of problems of cross-reactivity with Goa (see beginning ofResults section). The arrowheads point to the non-sensory epithelium,where anti-Gia1,2 gave some reaction, most likely background, as itcould not be blocked with excess antigenic peptide. Areas labeled 1, 2,19, and 20 (I) consist mainly of transitional epithelium (Figs. 6B and8). Upper case indicators in A and I: D, dorsal; L, lateral; V, ventral;M, medial. Scale bars 5 100 mm.

procedures described below were performed in accordancewith Federal and NIH animal use guidelines andinstitution-approved protocols.

Antibodies and peptides

All antibodies were rabbit polyclonal IgGs, unless men-tioned specifically. Antibodies used to localize Gia2, Goa,and TRP2 in the VNO receptor microvilli are listed inTable 1. Antibodies to the same proteins that did not workwell, antibodies to other proteins believed to be present inVNO receptor cells (Berghard and Buck, 1996; Wekesaand Anholt, 1997), and antibodies to MO signaling pro-teins used as controls (Menco, 1997b) are listed in Table 2.The antigenic peptides used to generate the Gia1,2 andGia1-3 antibodies differed from the corresponding region ofGoa in four and three amino acids, respectively (Watson

Fig. 3. Electron micrograph of the neurosensory epithelial surfaceof a conventionally fixed rat VNO embedded in Epon. The surfacecontains apices of receptor cell dendrites (asterisks) and of nearbysupporting cells (triangles). The latter tend to be somewhat moreelectron opaque than the former. The dendritic apices often show

clusters of centrioles (curved arrows). Thin microvilli sprouting fromthese apices are located close to the epithelial surface (straight ar-rows). The supporting cells have thicker and straighter microvilli(serpentine-shaped arrows), the course of which is roughly perpendic-ular to that of the receptor cell microvilli. Scale bar 5 1 mm.

Fig. 4. Antibodies to Gia1,2 (A; 1/10) and Goa (B; 1/10) bound tomicrovilli (straight arrows) of VNO receptor cells. The cell’s apices(large asterisks), including their membranes (marked by the straightlines parallel to the apices with large asterisks), as well as apices andmicrovilli of nearby receptor cells (small asterisks), and apices (trian-gles) and microvilli (serpentine-shaped arrows) of supporting cellsdisplay virtually no labeling. There is a gradient of reduced microvil-lar labeling in the direction of neighboring cells away from apices ofcells with labeled microvilli, suggesting microvillar dilution at somedistance from the cells from which these microvilli sprout (see alsoFigs. 8 and 9 and Tables 3 and 4). The curved arrows mark VNOreceptor cell centrioles. Apart from Figure 6D, all tissues in this andsubsequent figures were perfusion fixed with paraformaldehyde, rap-idly frozen, freeze substituted, and low temperature embedded inLowicryl K11M. The gold particles were 10 nm across. Scale bars 51 mm.


Figure 4

Fig. 5. VNO section as in Figure 4, but immunoreacted with TRP2(1/10). Although the apical membranes of the receptor cell dendrites(indicated by a short straight line parallel to the apex of one of thecells marked with a large asterisk; its centrioles are marked by acurved arrow) may have bound some of the antibodies, most IR wasfound on the receptor cell microvilli (straight arrows). In contrast to

the situation shown in Figure 4, where microvilli of only some of thereceptor were labeled, anti-TRP2 appears to have labeled the mi-crovilli of all the receptor cells (large asterisks; see also Fig. 11).Supporting cell apical (triangle) structures, including microvilli(serpentine-shaped arrow), were TRP2(-). The gold particles were 15nm across. Scale bar 5 1 mm.

and Arkinstall, 1994). Antibody and peptide dilutions arelisted in the appropriate figure captions.

Immunoblots: tissue preparation

Twenty rats were anesthetized deeply with 85 mg/kgsodium pentobarbital (Anthony Products, Arcadia, CA),intraperitoneally (ip). Dissected VNO tissues were frozenrapidly in liquid nitrogen and stored at 280°C. For themembrane preparation (Mishra, 1986), thawed tissueswere homogenized in 50 mM Tris (pH 7.4), 0.5 mM phe-nylmethylsulfonyl fluoride (PMSF), 1 mM p-tosyl-L-arginine methyl ester (TAME), 1 mM dithiothreitol (DTT),320 mM sucrose, 1 mM ethylenediamine tetra-acetic acid(EDTA), and 10 mM sodium metavanadate, by using aTissumizert homogenizer (Tekmar, Cincinnati, OH). Thesample was then centrifuged at 550 3 g for 5 minutes at4°C with a J2-21 centrifuge (Beckman, Palo Alto, CA). Thepellet was discarded. The supernatant was centrifuged at23,000 3 g for 15 minutes at 4°C. The 23,000 3 g pelletwas resuspended. The pellet and supernatant were ali-quoted (100 ml). The protein content of each was deter-mined by using the Bradford (1976) method for the super-natant (3.3 mg/ml) and pellet (1.2 mg/ml). The aliquotedsamples were then stored at 280°C for immunoblottingassays.

Immunoblots

After 10% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), samples were transferred topolyvinylidene fluoride (PVDF) membranes (Bio-Rad,Hercules, CA; Harlow and Lane, 1988). Transfers wereblocked with 5% nonfat dry milk (1 hour). IR was assessedwith secondary peroxidase-conjugated goat-anti-rabbit(GAR) IgG (1:5,000; Jackson ImmunoResearch Labs.,West Grove, PA), chemiluminescence reagent (NEN, Bos-ton, MA), and Kaleidoscope prestained standards (Bio-Rad) for molecular weights.

LM: tissue preparation

Deeply anesthetized adult rats were perfused transcar-dially with freshly prepared phosphate-buffered saline(PBS), pH 7.0, followed by Bouin’s fixative. The heads,trimmed of lower jaws, teeth, soft palates, skin, and mus-cle, were postfixed in fresh fixative overnight at 4°C,rinsed several times in 50% alcohol, decalcified in RDOt(Apex Engineering, Plainfield, IL), rinsed in running wa-ter, dehydrated through ethanol, cleared in Histosolt (Na-tional Diagnostics, Atlanta, GA), and embedded in Para-plastt (Sherwood Medical, St. Louis, MO). Sections(coronal, 10 mm) through the VNO and AOB weremounted on silanated slides (slides coated with 39-aminopropyl triethoxy silane, Sigma, St. Louis).

LM: immunocytochemistry

Deparaffinized (xylene) sections were rehydrated in agraded series of ethanol, preincubated as directed at roomtemperature, and incubated with the affinity-purified an-tibodies at appropriate dilutions in PBS for 2 hours at37°C. IR was visualized by using the rabbit VectastainABC Elitet kit (Vector, Burlingame, CA) with diamino-benzidine as the chromogenic agent.

EM: conventional methods

Deeply anesthetized rats were perfused with Kar-novsky’s (1965) aldehyde fixative in 0.1 M sodium cacody-

late, pH 7.4. Dissected VNOs were further fixed (2 hours),rinsed, postfixed in 1% OsO4, block stained with 3% ura-nyl acetate (UAc) in 50% ethanol, dehydrated, infiltratedwith Epon (Polysciences, Warrington, PA) through pro-pylene oxide, and embedded in Epon. Poststained (3% UAcin distilled H2O) and counterstained (lead citrate; Reyn-olds, 1963) ultrathin sections (Leica/Reichert Ultracut S,Vienna, Austria) were examined in a JEM-100-CX IItransmission EM at 120 kV (JEOL, Tokyo, Japan).

EM: rapid freezing and freeze substitution

Some antibodies work better in fixed tissues, others inunfixed tissues (Menco, 1995). For the preparation of un-fixed tissue, animals were asphyxiated with CO2 (1,000pounds per square inch, 30–45 seconds, until there was noheartbeat). VNOs were frozen rapidly on a liquid nitrogen-cooled copper block (Gentleman Jim Quick-Freezing Sys-tem, Energy Beam Sciences, Agawam, MA; Phillips andBoyne, 1984). Other animals were perfusion fixed with 4%paraformaldehyde 1 0.15 mM CaCl2 in 100 mM Soren-son’s phosphate buffer, pH 7.2. VNOs were further fixedovernight at 4°C by using the same fixative but in 100 mMbicarbonate (pH 9.4), washed, cryoprotected in sucrose/glycerol mixtures, and frozen rapidly in liquid propane byusing the Gentleman Jim. Unfixed tissues were freezesubstituted (Leica/Reichert CS Auto) through acetone andfixed tissues through methanol (both at 280°C). The hy-drophilic methacrylate resin Lowicryl K11M (ChemischeWerke Lowi G.m.b.H., Waldkraiburg, Germany), whichpolymerizes with ultraviolet light at low temperatures,was used for infiltration (280°C to 260°C) and embedding(260°C to 125°C; all in the CS Auto). The entire proce-dure lasted about 1 month (Berod et al., 1981; Van Look-eren Campagne et al., 1991; Menco, 1995; Menco et al.,1997).

EM: postembedding immunocytochemistry

Sections to be used for immunocytochemistry weremounted on 300 mesh thin-bar hexagonal nickel Gildergrids (EM Sciences, Fort Washington, PA). Blocking (2–3hours) and antibody dilution was done with 0.1% acety-lated bovine serum albumin (Ac-BSA; EM Sciences;Leunissen, 1990) in Trizma-buffered saline, 10 mM Tris,500 mM NaCl, pH 8.0 (TBS). Incubation with primaryantibodies was done overnight at 4°C without intermedi-ary wash. Their binding was visualized with 10 or 15-nmgold particles conjugated to secondary goat-anti-rabbit(GAR) or rabbit-anti-goat (RAG) for polyclonal primaryantibodies and to goat-anti-mouse (GAM) for monoclonalantibodies (EM Sciences). Gold colloids were diluted in 20mM Tris, 150 mM NaCl, 0.1% Ac-BSA, pH 8.2, to anoptical density of about 0.10 at 520 nm (Menco et al.,1994).

Identification of cells in sequential sections

Most of the primary antibodies used were generated inthe same species, making double labeling of these anti-bodies problematic. To avoid questions of secondary anti-body cross-reactivity, immunocytochemistry for differentprimary antibodies was carried out on adjacent sections.Large composite micrographs were prepared of the sameregion of epithelium in adjacent serial sections that hadbeen immunoreacted with antibodies to either Gia2 or Goa.The same was done for each of these subunits and TRP2.


Figure 6

This allowed careful examination of matched regions ofVNO neuroepithelium in sequential sections. The compos-ites were delimited by the bars of the 300-mesh hexagonalgrids holding the sections. These areas could be up to 73mm in diameter, the maximum hole width of these grids(EM Sciences Catalogue XIII, p. 61). Sets of compositesconsisted of up to 80 individual micrographs (4 sections,20 exposures). Low power magnifications (1003 to 2003)served as a guide for matching of regions in the higherpower micrographs. Successive regions, labeled 1 through9 and 19 and 20 (see Fig. 2I), are defined by the successiveoccurrence of the VNO neuroepithelial surface in the opengrid areas. Lowest and highest numbers mark the transi-tional epithelium closest to the non-sensory VNO epithe-lium. The highest number, 20, is selected arbitrarily be-cause it did not overlap in any of the sections with regionnumbers counting from the other side, marked 1.

Controls

Antibody specificity for the G-proteins and TRP2 wasverified by preabsorption with an excess of correspond-ing antigen in immunocytochemical controls. Also, thelabeling pattern of each antibody was a positive controlfor all others. Tissue controls were the non-sensory areaof the VNO, the sensory cilia and supporting cell mi-crovilli of the MO, and the non-sensory cilia and mi-crovilli of the nasal respiratory epithelium. Antibodiesto signaling proteins of MO olfactory cilia, applied toVNO, served as additional tissue controls (Table 2, note6). Negative controls consisted of PBS, and in the case ofpolyclonal antibodies, preimmune serum, or normalrabbit (NRS) or goat sera (NGS), all with the appropri-ate blocking solutions and all used instead of the pri-mary antibodies.

Data analysis

Densities were counted in microvillar tufts apical to thesensory cells from which the microvilli sprout, in suchtufts above neighboring sensory cells, and in the sur-rounding resin (Table 3). For this, a transparent plasticsheet with a grid divided in cm2 squares was placed over

each micrograph. Gold grains per cm2, recalculated permm2, reflecting the intensity of labeling of each antibody ineach region, were statistically analyzed by one-way anal-yses of variance (ANOVAs) by using Statview 4.0 on aMacintosh computer (Menco et al., 1997).

RESULTS

Verification of IR

For proper evaluation of the fine structural studies pre-sented here, it was necessary to verify in immunoblots andwith LM IR in VNO tissues for several of the antibodies(Tables 1, 2). Antibodies to Gia2 in the VNO that were usedby others (Shinohara et al., 1992; Jia and Halpern, 1996)were not available to us. A commercially available mono-clonal antibody to Gia2 (Li et al., 1995) did not work in ourstudy (Table 2). However, it labeled dendritic endings andthe microvilli of VNO receptor cells in another, pre-embedding, fine structural study (Matsuoka et al., 2001).Therefore, to examine IR for Gia2, two antibodies wereused, one to Gia1,2 and one to Gia1-3 (Table 1), the latterbeing used in the VNO by several others (Berghard andBuck, 1996; Murphy et al., 2001). In the AOB, the anteriorregion showed IR to Gia1,2, whereas the posterior portiondemonstrated IR to Goa. The IR patterns for those anti-bodies agree with previous findings (Shinohara et al.,1992; Halpern et al., 1995, 1998; Jia and Halpern, 1996;Jia et al., 1997; Sugai et al., 1997). In contrast, antibodiesto Gia1-3 bound equally well to the posterior, Goa(1), andanterior, Gia1,2(1), regions of the AOB, indicating that theGia1-3 antibodies displayed cross-reactivity for Goa (Aokiet al., 1992). This confirmed a suspicion based on EMobservations that suggested that the microvilli of all VNOreceptor cells immunoreacted with antibodies Gia1-3. Toavoid this problem of cross-reactivity, antibodies to Gia1,2were subsequently used mainly to demonstrate IR for Gia2in the VNO. TRP2 antibodies were only used for EM; blotand LM IR of VNO epithelia for these antibodies is shownelsewhere (Liman et al., 1999).

Immunoblots

Western blots of membrane preparations of VNO epi-thelia were tested for the presence of the a-subunits of theGo and Gi proteins (Tables 1, 2, Fig. 1). Bands of appro-priate molecular weights were observed for all antibodiestested, except for those to Gia1. The band at 39–40 kDa isconsistent with the molecular weight of Goa (Fig. 1A).Bands at molecular weights of 40–41 kDa are consistentwith the molecular weights of Gia2 and Gia3 (Figs. 1B,C;Watson and Arkinstall, 1994; for Gia1-3 in the VNO, seealso Murphy et al., 2001). No band was obtained for Gia1(Fig. 1D), indicating that Gia1 is not present in the VNO.Thus, any Gi-type labeling in the LM and EM studiesbelow must be due to the presence of Gia2 or Gia3. Com-pany specifications indicated that the Gia1-3 and the Gia1,2antibodies label Gia2, Gi2 being the G-protein of the Gi(1)VNO receptor cells (Berghard and Buck, 1996). This val-idated the use here of the anti-Gia1-3, but especially anti-Gia1,2 (the Gia1-3 antibodies were of limited use because ofthe problems outlined above). The blots in Figures 1A-Calso show that more G-protein IR was associated with thepellets than with supernatants. Extra bands in both pel-lets and supernatants for the antibodies to Gia1,2 andGia1-3 may reflect degradation or aggregation products or

Fig. 6. Absence of IR in non-sensory VNO cells (A-C) and labelingpatterns in unfixed tissues (D). Cilia (large asterisk in A) and mi-crovilli (small asterisk A) of VNO ciliated non-sensory cells and mi-crovilli of cells in the transitional region to the non-sensory epithe-lium (arrow in B; region 1 in Fig. 2I) did not label with anti-TRP2(1/10). The same was true for antibodies to the G-proteins. C: Thetransitional region (near region 2 in Fig. 2I) contained some cells thatresemble MO receptor cells in their apices (triangle). However, thecilia of these cells (large arrowhead) bound neither antibodies to VNOnor those to MO signaling molecules (here: anti-Gia1-3, 1/12;) as wastrue for apices (small asterisk) and microvilli (small arrowhead) ofVNO-supporting cells. VNO receptor cells (large asterisk) in this areahad Gia2(1) microvilli (arrow), but none had microvilli that wereGoa(1). The apex from which the microvilli sprout also showed someIR (large asterisk). D: Antibodies to Gia1,2 (1/10) bound to the mi-crovilli of a VNO receptor cell (straight arrows) in unfixed and non-cryoprotected tissues. The apex of the receptor cell (large asterisk) aswell as apices (small asterisk) and microvilli of surrounding support-ing cells displayed no labeling. The border between the microvilloussurface and the feature-less resin (wide arrow) is an artifact caused byslamming the tissue on the cold copper block used for cryofixation. Thegold particles in A,B were 15 nm across and those in C,D were 10 nm.Scale bars 5 1 mm.


Figure 7

nonspecific impurities present in the antibody prepara-tions.

LM

IR for Gia1-3 (Figs. 2A,D), Gia1,2 (Figs. 2B,E), and Goa

(Figs. 2C,F) was restricted to the epithelial surface andaxons (Berghard and Buck, 1996). Anti-Gia1,2 also labeledcell apices in the non-sensory VNO area (Fig. 2E); immu-noabsorption controls showed this to be nonspecific andthis labeling was not seen with EM. As seen in the immu-noblots, anti-Gia1 showed no IR over control levels (Figs.2G,H). Figure 2I serves as a map relating the LM surveyoutlined here with the EM topographical study further onin this paper (see Figs. 8–10).

EM: the luminal area of the rat VNO

Although the ultrastructural morphology of the ratVNO epithelial surface with conventional EM is well es-tablished (Vaccarezza et al., 1981; Mendoza and Szabo,1988; Garossa and Coca, 1991; Johnson et al., 1993), wehighlight some aspects that help in the interpretation ofthe fine structural IR data. Receptor cell apices are widerand appear more electron lucent than those of neighboringsupporting cells. The apices of both cell types bear mi-crovilli. Those of receptor cells are 0.03–0.06 mm wide and3–5 mm long (Table 4). They tie parallel to the epithelialsurface, and close to it. Supporting cell microvilli arethicker (0.1–0.2 mm) and shorter (2.5–3.5 mm), tie perpen-dicular to the receptor cell microvilli, and their tips extendabove the layer that contains the latter (Fig. 3, see alsoFig. 12).

EM: localization of signaling proteins

With EM, the affinity-purified antibodies (Table 1) toGia1,2 (Fig. 4A), Goa (Fig. 4B), and TRP2 (Fig. 5) showedhighly specific IR of the microvilli of VNO receptor cells.Anti-TRP2 also shows some IR in apical membranes of thereceptor cell dendrites in between the microvilli (Fig. 5).Otherwise, labeling in other parts of the receptor cellapices was much lower or absent for all antibodies. Recep-tor cell axons, not further considered here, were also pos-itive for Gia1,2, Gia1-3, and Goa, although less so than themicrovilli. Axons were TRP2-(-).

IR was absent from the microvilli and cilia of almost allother cell types examined. These include the microvilli ofVNO supporting cells (Figs. 4, 5; see also Figs. 8, 9) and ofminor populations of different-appearing VNO microvil-lous cells (not shown), cilia and microvilli of the VNOnon-sensory epithelium (Fig. 6A), the microvilli in thetransitional area between the sensory and non-sensoryepithelium (Fig. 6B), and nasal respiratory epithelia. Thetransitional area contains some ciliated cells, the apices ofwhich resemble those of ciliated receptor cells of the MO.In contrast to the microvilli of nearby VNO sensory cells(see Fig. 8), the apices and cilia of the ciliated cells neithershowed IR for the VNO signaling molecules explored here(Fig. 6C) nor IR for the signaling molecules of the MOreceptor cell cilia (Fig. 7E), such as Golfa and type III AC(Menco et al., 1994; Table 2). Antibodies to the Gi subunitslabeled receptor cell microvilli in fixed (Figs. 4A, 6C, 8A,9A,B; see also Fig. 11A) and unfixed freeze-substitutedtissues (Fig. 6D); those to Goa (Figs. 4B, 8B, 9C,D; see alsoFig. 11B) and TRP2 (Fig. 5; see also Figs. 11C,D) onlylabeled microvilli in fixed tissues.

Omission of primary antibodies from the reaction mix-ture gave no labeling in either LM (Figs. 2G,H) or EMpreparations. Preabsorption of Gia1,2, Gia1-3, Goa, andTRP2 antibodies with their respective antigenic peptidessuppressed, both at LM and EM levels, IR to background,as shown here for Gia1,2 (Fig. 7A) and TRP2 (Fig. 7B) inVNO receptor cell microvilli (compare with Figs. 4A and 5,respectively). Antibodies to Gia2 and TRP2 did not immu-noreact with MO sensory cell cilia and the microvilli ofsupporting and other MO microvillous cells (Figs. 7C,D).Gia1 labeled diffusely MO epithelia (not shown; Sinnara-jah et al., 1998). Goa IR was somewhat prominent in theMO, but its labeling pattern was different from that seenin the VNO. In the VNO, the microvilli of a subpopulationof receptor cells were specifically Goa(1). In the MO, theapical cytoplasm of supporting cells, but not their mi-crovilli, was labeled (Menco et al., 1994). None of theantibodies to MO signal-transduction proteins labeledVNO receptor cell microvilli. Besides antibodies to Golfa(Figs. 7E,F), these included antibodies to Gsa, type III AC(Menco et al., 1994), OCNC1 (Matsuzaki et al., 1999),Gaq/11 (DellaCorte et al., 1996), and inositol 1,4,5-trisphosphate (IP3) receptors (Cunningham et al., 1994;Kalinoski et al., 1994; Table 2).

Several antibodies did not work well. Non-purified an-tibodies to the G-proteins and olfactory marker protein(OMP; Table 2) labeled VNO, supporting cell microvilliinstead of receptor cell microvilli, as was the case withNRS and NGS. Hence, this labeling pattern was consid-ered to be nonspecific. Antibodies to other proteins be-lieved to be involved in VNO signaling, type II AC(Berghard and Buck, 1996), Gaq11, and IP3 receptors(Wekesa and Anholt, 1997; Sasaki et al., 1999), gave neg-ative or inconclusive (scattered labeling in no apparentpattern) IR results (Table 2).

Epithelial topography

To demonstrate localization of tufts of Gia2(1) andGoa(1) microvilli belonging to nearby receptor cells alongthe VNO sensory surface, two sets of serial sections areincluded. Figure 8 shows such differential labeling for anarea near the transition from non-sensory to sensory epi-thelium (region 2, Fig. 2I). Figure 9 shows this for a morecentrally located area (region 7, Fig. 2I). The very initial

Fig. 7. Immunoabsorption (A,B) and tissue controls (C-F). A:VNO labeled with anti-Gia1,2 (1/10) preabsorbed with a 10-fold molarexcess of the antigenic peptide (A; Gia1,2-p) or with anti-TRP2 (1/10)preabsorbed with a thousand-fold molar excess of the antigenic pep-tide (B; TRP2-p) suppressed dramatically IR of the receptor cell mi-crovilli (arrows; compare with Figs. 4A and 5). The molecular weightof the TRP2 peptide was 40 kDa and that of the antibody approxi-mately 160 kDa (Liman et al., 1999). C,D: All MO epithelial surfacestructures were TRP2(-) (1/10). This includes receptor cells and theircilia (C) and the apex (asterisk in D) and microvilli (arrow in D) of amicrovillous cell that differed from regular supporting cells (D; Carret al., 1991; Menco, 1994). E,F: MO (E) and VNO epithelial surface (F)that were immunoreacted with antibodies to the MO G-protein sub-unit Golfa (1/5). IR is prominent in distal parts of the MO cilia (arrowin E) and is sparse and scattered in the VNO epithelial surface, alsoat the level of receptor cell microvilli (arrow in F). In A-F: Largeasterisks, receptor cell apices; triangles, supporting cell apices;serpentine-shaped arrows, supporting cell microvilli; small asterisksin C and E, proximal parts of olfactory cilia. Gold particles were 10 nm(A,E,F) and 15 nm (B,C,D) across. Scale bars 5 1 mm.


Fig. 8. Composites of serial sections, immunoreacted with anti-bodies to Gia1,2 (A; 1/5) and Goa (B,C; 1/10), in the VNO neuroepithe-lial surface in an area of transition to the non-sensory area of the VNO(region 2 in Fig. 2I). The low power micrograph (B) presents a largerarea of the section depicted in C. Matching indicators mark the samestructures. iA-iE mark apices of individual cells with Gia1,2(1) mi-crovilli and oA marks the one individual cell with Goa(1) microvilli.

Sensory cell apices, including their membranes (e.g., the area parallelto the short straight line of the apex of the cell labeled oA), displayedmuch less labeling than the cells’ microvilli. Tufts of labeled microvilliare marked with straight arrows, surrounding supporting cells aremarked with large arrowheads, and their unlabeled microvilli withserpentine-shaped arrows. The gold particles were 10 nm across in Aand 15 nm in B,C. Scale bars 5 1 mm in A,C, 10 mm in B.

Figure 8 (Continued)


Fig

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part of the transition zone between the VNO sensory andnon-sensory epithelium had no cells that reacted with theantibodies (Fig. 6B). IR only became apparent about half-way within transitional regions 1 or 20 (Fig. 2I), where themicrovilli of most receptor cells were Gia1,2(1). That is,along a stretch of approximately 20 mm, the microvilli ofone of six cells were Goa(1) (cell oA in Figs. 8A,C). Incontrast, more central regions had about equal numbers ofcells with Gia1,2(1) and Goa(1) microvilli (Figs. 9A–E).Along a 30 mm stretch, there were four cells with Gia1,2(1)microvilli and three with Goa(1) microvilli (Fig. 9E). Sum-marized over four animals, Figure 10 shows that in thetransition zone near the non-sensory VNO epithelium (re-gions 1–3, and 19 and 20 in Fig. 2I), significantly morereceptor cells had Gia2(1) microvilli than had Goa(1) mi-crovilli (P , 0.05). In the more central region, these num-bers were about equal (regions 4–9 in Fig. 2I). Total den-sities of all receptor cells expressing microvilli—bothGia2(1) and Goa(1)—were 200–300 cells per mm or 4 to9 3 106 cells per cm2 epithelium.

The immunocytochemical distinction in receptor cells isnot accompanied by differences in the morphological ap-pearance of the apices of the cells. Apices of receptor cellsbearing Gia2(1) microvilli were indistinguishable from

those bearing Goa(1) microvilli. Features like widths ofthe apices and of microvillar tufts and the presence ofcentrioles inside the cells were the same for both types ofsensory cells. However, the microvilli of one cell extendover the surface of neighboring cells, where labeling in-tensities are diluted in the microvillar tufts of these neigh-boring cells (Figs. 4, 8, and 9; Table 4). By using serialsections and a semiquantitative analysis, we were able todistinguish between cells that contribute to the microvil-lar labeling for Gia2 or Goa, despite their morphologicalsimilarities. Taking the labeling density of microvillartufts apical to the cell from which the microvilli sprout as100% reveals that the labeling density of these microvilli,when they extend into the microvillar tufts above apices ofneighboring sensory cells, is 30–50% (Table 3).

In contrast to Gia1,2 and Goa, which appeared to havelabeled the microvilli of two distinct types of receptor cells,the microvilli of both of these receptor cells, thus thosewith Gia1,2(1) and those with Goa(1) microvilli, areTRP2(1), also in the transition zone. Furthermore, themicrovilli of both cell types display a similar intensity oflabeling for TRP2 (Fig. 11).

Fig. 10. Histogram showing that the transition zone between thesensory and non-sensory VNO epithelium (regions 1-3 and 19-20 inFig. 2I) has significantly (P , 0.05; asterisks) more receptor cells withGia2(1) microvilli (dark gray) than with Goa(1) microvilli (light gray).More centrally, these numbers are about equal. Values in region 3differed significantly (bullet, P , 0.05) from both, those with more andthose with fewer, labeled cells. The results are based on four animals.Bars, standard errors.

TABLE 3. Semiquantitative Evaluations on the UltrastructuralLocalization of Gia2 and Goa as Assessed by Densities of Gold Particles inMicrovillar Tufts Above VNO Sensory Cells That Contain the Particular

Antigen, Above Neighboring Cells That Do Not Contain the Antigen,and in the Embedding Resin

Antibody to: Gia2(1) cell Goa(1) cell Resin

Gia1,2 (15 nm)1 8.5 6 4.9 (53)2 3.0 6 2.1 (42) 0.7 6 0.6 (17)100%3 35% 8%Labeled cell Neighboring cell

Gia1,2 (10 nm)1 13.2 6 6.0 (32) 5.8 6 2.6 (26) 0.6 6 0.8 (8)100% 44% 5%Labeled cell Neighboring cell

Goa (15 nm) 3.7 6 2.8 (63) 9.6 6 6.3 (67) 0.5 6 0.5 (26)39% 100% 5%Neighboring cell Labeled cell

1Primary antibodies to Gia1,2 were used to localize Gia2. Two sizes of gold grains wereused for the secondary antibodies, 10 and 15 nm, to localize Gia2.2Values 6 standard deviations (number of cells for which the values were determinedwithin parentheses) are given in density of gold grains per mm2 microvillous tuft abovea designated cell. Structure-devoid resin served as the control area for background.Within rows, all values differed significantly (P , 0.0001). Absolute values withincolumns cannot be compared, as each column depicts a different antibody.3Density values above the cells targeted by the antibodies are highest, 100%. Othervalues in the same rows are given as a percentage of this value. In other words, in thefirst row, the density of Gia2(1) gold grains apical to neighboring Goa(1) cells is 35%compared with that in microvillous regions apical to cells that express the Gia2(1)protein. This spread is caused by microvillous tufts being wider than cell apices (seeTable 4, third column).

TABLE 4. Widths of Apices and of Microvillous Tufts ofRat VNO Sensory Cells

Sensory cellapices

Sensory cellmicrovillous

tufts

Average widths ofthe microvilloustufts relative to

the averagediameters of the

apices of the cells(percentages)

Gia2(1) cells 3.3 6 1.5 (134)1 5.4 6 2.1 (48)1,2 160%Goa(1) cells 3.2 6 1.2 (80) 7.0 6 2.5 (38) 219%

1Values are given in mm 6 standard deviations, with the number of cells for which thevalues were determined in parentheses. Within rows, values differed significantly (P ,0.0001).2Widths of tufts are based on the length of microvilli of individual cells that could befollowed in single sections and on the extent of labeling in serial sections of receptor cellmicrovilli when these cells were sufficiently spatially separated to identify their mi-crovilli as originating from individual cells.

Fig. 9 (Overleaf). Composites of serial sections, immunoreactedwith antibodies to Gia1,2 (A,B; 1/5) and Goa (C-E; 1/10), through a VNOneuroepithelial surface area situated more centrally (region 7 in Fig.2I) than the area shown in Figure 8. The low power micrograph (D)presents a larger area of the section depicted in E. Matching indica-tors mark the same structures. iA, iB, and oA in A-C and E or iA-iD andoA-oC in D mark the apices of individual cells with Gia1,2(1), respec-tively, Goa(1) microvilli. These apices display much less labeling thantheir microvilli. Absence of labeling includes apical membranes (e.g.,the areas parallel to the short straight lines of the apices of the cellslabeled iB in A and B and those of the cells labeled oA in C and E).Tufts of labeled microvilli are marked with straight arrows. Support-ing cells are marked with large arrowheads, their unlabeled microvilliwith serpentine-shaped arrows. The gold particles were 10 nm (A,B)and 15 nm across (C-E). Scale bars 5 1 mm in A-C and E, 10 mm in D.


DISCUSSION

This study demonstrates clearly that VNO receptor cellmicrovilli are highly specialized for sensory signaling.Other fine structural characterizations of VNO receptorcells have shown that apices and microvilli of rat VNOreceptor cells contain Gia2 and Goa (Matsuoka et al., 2001).However, this study shows that the microvilli of VNOreceptor cells displayed a much more prominent IR fora-subunits of the signaling G-proteins Gi2 and Go and forTRP2 than any other component of the VNO receptor cellapices (summarized in Fig. 12). This situation is compa-rable to the situation in the MO, where signaling mole-cules are especially abundant in the long distal parts ofreceptor cell cilia (summarized in Menco, 1997a). It hadbeen established that the membrane appearances of MOcilia and VNO microvilli, as reflected in the densities of

intramembranous particles, are alike (Breipohl et al.,1982; Menco, 1992). This is true despite differences in thecytoskeletal substructure (Vaccarezza et al., 1981) and thelength of these subcellular hairlets (rodent VNO receptorcell microvilli are 5–10 mm long [this paper; Naguro andBreipohl, 1982]; rodent MO receptor cell cilia are 50–60mm long [Seifert, 1970]). The similarity in the appearanceof the membranes of the two types of cellular hairletssuggested that both structures subserve a similar func-tion, i.e., the subcellular sites of chemosensory signal in-put. As there is virtually no other information on thespecial properties of VNO receptor cell microvilli, the newinformation may provide the strongest evidence to datethat these microvilli are adapted specifically for their che-moreceptive function. Consequently, the data provide cel-lular insight important to understand the complex physi-

Fig. 11. Two areas (A,C, and B,D) in VNO serial sections from thesame rat that had been immunoreacted with affinity-purified antibod-ies to Gia1,2 (A; 1/10), Goa (B; 1/10), and TRP2 channels (C, D; 1/10).Microvilli in corresponding serial sections (arrows) of both Gia1,2(1)(A) and Goa(1) receptor cells (B) were also TRP2(1) (C and D, respec-

tively). Membranes of receptor cell apices (asterisks) reacted some-what with the antibodies to TRP2, but not with those to theG-proteins. Apices (triangle) and microvilli (serpentine-shaped ar-rows) of surrounding supporting cells did not label with any of theantibodies. The gold particles were 15 nm across. Scale bars 5 1 mm.


ology and biochemistry of VNO signaling (Døving andTrotier, 1998; Liman, 1996, 2001; Keverne, 1999; Dulac,2000; Holy et al., 2000; Leinders-Zufall et al., 2000).

Cells with Gia2(1) and Goa(1) microvillicoexist within the VNO neuroepithelium

Until now, the pattern of the differential distributions ofGia2(1) and Goa(1) VNO receptor cells at the level of thecells’ microvilli remained unclear. LM immunolabelingpatterns of rodent VNO receptor cells throughout thesecells led Jia and Halpern (1996) to postulate that there aretwo types of these cells, Goa(1) and Gia2(1). This wasconfirmed by in situ hybridization (Berghard and Buck,1996). Later studies demonstrated that these receptor cellpopulations differ from each other in other importantchemical aspects. These differences include the nature ofthe putative VNO odorant receptors that the cells express(Herrada and Dulac, 1997; Matsunami and Buck, 1997;Ryba and Tirindelli, 1997) and the nature of some othermolecules that may participate in VNO signaling, such asphosphodiesterases (Lau and Cherry, 2000). The samewas true for several molecules that participate in thedevelopment of the two populations (Kishimoto et al.,1993; von Campenhausen et al., 1997; Halpern et al.,1998). Use of serial sections yielded a sufficiently largenumber of spatially separated cells now, to make the pointthat the antibodies to Gia2 and Goa labeled microvilli ofdifferent VNO receptor cells. The fact that in the transi-tion zone to the non-sensory VNO epithelium, the VNOepithelial surface contains primarily cells with Gia1,2(1)microvilli helped to strengthen this case. These findingsare important to understand the putative roles of Gia2 andGoa in VNO signaling.

Mechanism of gating of TRP2

TRP2 is a most suitable candidate for the current gen-erating channel in all VNO receptor cells. In situ hybrid-ization studies showed that Gia2(1) and Goa(1) VNO re-ceptor cells express TRP2 (Liman et al., 1999). This studyadded that this is especially true at the sites of potentialinput, receptor cell microvilli; microvilli of receptor cellsthat are Gia2(1) and those that are Goa(1) are TRP2(1).TRP2 is a member of a class of ion channels that have beensuggested by some (Vannier et al., 1999) to mediate Ca21

influx across the plasma membrane in response to releaseof Ca21 from intracellular stores. However, other re-searchers (Liman et al., 1999) have proposed that thesechannels open in response to a phospholipid signalingcascade in a Ca21 store-independent manner. The ultra-structural data presented here show that TRP2 is mostabundant in sensory microvilli, structures that are un-likely to have an intracellular Ca21-containing membra-nous compartment, such as endoplasmic reticulum.Therefore, it is improbable that TRP2 is gated in responseto the release of Ca21 from an intracellular store (Vannieret al., 1999). Although Ca21 stores may be present in thedendritic apex, communicating at a distance to the TRP2channels in the microvilli, it is more probable that TRP2 isgated by a second messenger, as is true for the TRPchannels involved in the phototransduction cascade ofDrosophila melanogaster (Acharya et al., 1997; Chyb etal., 1999). In the VNO receptor cell microvilli, this mes-senger may be generated by activation of chemosensoryreceptors. Elevation of diacylglycerol (DAG) and/or IP3,resulting from a PLC-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate, conceivably leads to opening ofTRP2 channels (Liman et al., 1999; Holy et al., 2000).

Other signaling proteins and other species

In rodents, there is evidence that type II (Berghard andBuck, 1996) and/or type VI AC (Rossler et al., 2000) andan OCNC2 channel (Berghard et al., 1996) also play a rolein VNO sensory signaling. It is unclear whether thesemolecules mediate and/or modulate transduction togetherwith the molecules in the receptor cell microvilli, orwhether they are involved in other, e.g., non-sensory, pro-cesses. Also, the VNO of other species may, at least inpart, make use of a somewhat different array of signalingmolecules. For example, in the goat, all VNO signalingtentatively involves Gia2 (Takigami et al., 2000), whereasGqa may play a role in the pig (Wekesa and Anholt, 1997).

Ciliated cells in the VNO and atypicalmicrovillous cells in the MO and VNO

The cilia of the sparse ciliated cells that resemble MOreceptor cells in their apices (Fig. 6C; Adams andWiekamp, 1984) and that are present in the transitionarea between the sensory and the non-sensory regions ofthe VNO did not immunoreact for any of the MO or VNOsignaling molecules. Therefore, these ciliated cells may bethe same as those found in the non-sensory VNO. Simi-larly, the microvilli of some atypical microvillous cells inthe VNO (not shown) and MO (Fig. 7D; Carr et al., 1991;Menco, 1994) did not immunoreact with antibodies toVNO or MO signaling proteins, making it unlikely thatthese are VNO (or MO)-type receptor cells.

Fig. 12. Summary diagram of the major sites of localization ofVNO signal transduction components. IR for all signaling proteins:Gia2 (visualized with antibodies to Gia1,2), Goa, and TRP2 channels areparticularly concentrated in receptor cell microvilli. Scale bar 5 1 mm.


Topography

There is a differential distribution of receptor cells withGia2(1) and Goa(1) microvilli along the VNO epithelialsurface (Fig. 10). This surface topographic patterning re-sembles somewhat the developmentally expressed topo-graphic pattern in the MO (Menco and Jackson, 1997).The latter parallels roughly four zones in which mostputative MO odorant receptors are located (Ressler et al.,1993; Vassar et al., 1993). The current data, together withresults of in situ hybridization studies (Herrada and Du-lac, 1997), suggest that three such zones may be present inthe VNO: the dorsal and ventral transitional areas withmainly cells with Gia2(1) microvilli coincide roughly withthe areas where most proliferation is concentrated(Berghard and Buck, 1996; Weiler et al., 1999). TheGia2(1) cells may be those that are mainly mature in thisarea. Alternatively, they may comprise a special popula-tion of cells in this region. A recent developmental studysuggests that the latter is the case indeed (Giacobini et al.,2000). In the more central parts of the VNO neuroepithe-lium, where numbers of Gia2(1) and Goa(1) cells are aboutequal, our data agree with those of Matsuoka et al. (2001).

Although VNO receptor cell apices are about 1.5–2times (Table 4) as wide as those of MO receptor cells,apices of VNO supporting cells are narrower (Fig. 3). Thismay be the reason why overall densities of VNO microvil-lous receptor cells, summated over Gia2(1) and Goa(1)ones, resemble those of MO ciliated receptor cells (Menco,1983).

Some technical and cautionary notes

Labeling restricted to VNO microvillar regions noted inthis study is consistent with some earlier LM studies(Berghard and Buck, 1996; Murphy et al., 2001), includingobservations on microvilli of singly dissociated VNO neu-rons, that demonstrated that these can be both Gia2(1)and TRP2(1) (Liman et al., 1999). However, the new datadiffered somewhat from those reported in other LM (Halp-ern et al., 1995; Jia and Halpern, 1996; Jia et al., 1997)and EM studies (Matsuoka et al., 2001). This is becausethe other investigators used antibodies to Goa and Gia2,which labeled additional parts of VNO receptor cell apices.The differences may be due to variances in tissue prepa-ration and/or antibody source. The antibody-antigen inter-action can be altered with different tissue preparationconditions (Griffiths, 1993; Menco, 1995; Webster, 2000).Therefore, one has to be careful when making assertionson a single labeling condition. For example, besides thedifferences noted above, we also encountered nonspecificlabeling (labeling of VNO supporting cell microvilli in thecase of non-purified antibodies, see also subscript 12 inTable 2), cross-reactivity (antibodies to Gia1-3 also labeledGoa), and differential labeling under various fixation con-ditions (antibodies to Gia1,2 and Gia1-3 worked well in fixedand unfixed tissues whereas those to Goa and TRP2 onlyworked in fixed tissues). Finally, each gold particle couldhave bound to more proteins and/or the gold particlescould not access all receptors. Actual receptor densitiescould even be about 10 times higher than the densityvalues presented in Table 3 (Phillips and Bridgman,1991). For that reason, the values in Table 3 merely serveas a guide for minimum numbers of labeled proteins.

Conclusions

First, this study gave the most pertinent evidence todate that VNO receptor cell microvilli are enriched specif-ically in the putative signaling molecules Gia2, Goa, andTRP2, providing important support for the role of thesemolecules in VNO sensory transduction. Second, there aretwo types of receptor cells. One of these has Gia2(1) mi-crovilli, the other has Goa(1) microvilli. Otherwise, thesereceptor cells are morphologically indistinguishable intheir apices. Third, the microvilli of both receptor celltypes are TRP2(1). Fourth, a zonal distribution of thesereceptor cell types also occurs. Many more cells withGia2(1) microvilli than cells with Goa(1) microvilli arepresent in the transition zone near the non-sensory VNOepithelium.

ACKNOWLEDGMENTS

We acknowledge the help of Drs. Q. Tian Wang and R.A.Holmgren, Northwestern University, Department of Bio-chemistry, Molecular Biology and Cell Biology in the prep-aration of the immunoblots. Gene Minner is thanked forphotographic assistance.

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